During sepsis in either humans or in animal models, activation of innate immune cells by endotoxin leads to systemic inflammation via overproduction of proinflammatory cytokines resulting in acute lung injury (ALI). During this process, several pathways are operative in endothelial cells including, oxidative stress, intracellular Ca2+ overload and organelle dysfunction and endothelial cell death. Despite substantial information regarding the underlying molecular mechanisms that lead to endotoxin-induced ALI, several elements in the pathway remain to be identified. Identification of the molecular components in ALI is of critical importance in order to understand the progression of ALI pathogenesis and to identify potential targets for treatment of the disease. Both paracrine and autocrine-derived oxidants are known to modulate the redox biology of the endothelial cells. Our recent studies demonstrate that both immune cell- and autocrine-derived oxidants activate pulmonary endothelial Ca2+ signaling, while blockade of oxidant production inhibits Ca2+ signaling induced proinflammatory responses. Further, we have recently demonstrated that the ER resident Ca2+ sensor STIM1 is also a ROS target. Hence, oxidation of STIM1 facilitates activation of the plasma membrane Ca2+ channel Orai1, thereby elevating cytosolic Ca2+ levels and regulation of gene transcription. The ability of STIM-operated channels to elevate intracellular Ca2+ levels and induce transcriptional activity prompted us to investigate its role in inflammation caused by endotoxin. Our hypothesis for work proposed in this application is that vascular inflammation results from LPS-induced ROS overproduction which leads to perturbation of Ca2+ signaling, disruption of mitochondrial function and energy metabolism, loss of vascular tone and vascular integrity. The specific aims of this project are: 1) Characterize the role of ROS-induced Ca2+ signaling in endothelial cell activation and inflammation; and 2) To examine whether targeting the CRAC channel components control vascular inflammatory responses. Our goal is to characterize novel new mechanisms involving ROS, Ca2+ and related intracellular events that modulate gene transcription in endothelial cells after either in vitro or in vivo exposure to endotoxin which could contribute to development of sepsis. These investigations will provide new insights into the fundamental mechanisms that modulate endothelial function and contribute to the ALI pathogenesis.